Vassar Alzheimer's Research & Therapy (2014) 6:89 DOI 10.1186/s13195-014-0089-7

REVIEW

BACE1 inhibitor drugs in clinical trials forAlzheimer’s diseaseRobert Vassar

Abstract

β-site amyloid precursor protein cleaving enzyme 1 (BACE1) is the β-secretase enzyme required for the productionof the neurotoxic β-amyloid (Aβ) peptide that is widely considered to have a crucial early role in the etiology ofAlzheimer’s disease (AD). As a result, BACE1 has emerged as a prime drug target for reducing the levels of Aβ inthe AD brain, and the development of BACE1 inhibitors as therapeutic agents is being vigorously pursued. It hasproven difficult for the pharmaceutical industry to design BACE1 inhibitor drugs that pass the blood–brain barrier,however this challenge has recently been met and BACE1 inhibitors are now in human clinical trials to test forsafety and efficacy in AD patients and individuals with pre-symptomatic AD. Initial results suggest that some ofthese BACE1 inhibitor drugs are well tolerated, although others have dropped out because of toxicity and it is stilltoo early to know whether any will be effective for the prevention or treatment of AD. Additionally, based on newlyidentified BACE1 substrates and phenotypes of mice that lack BACE1, concerns have emerged about potentialmechanism-based side effects of BACE1 inhibitor drugs with chronic administration. It is hoped that a therapeuticwindow can be achieved that balances safety and efficacy. This review summarizes the current state of progress inthe development of BACE1 inhibitor drugs and the evaluation of their therapeutic potential for AD.

IntroductionThe role of β-amyloid in Alzheimer’s diseaseThe extracellular accumulation of amyloid plaques com-posed of the β-amyloid (Aβ) peptide represents one of thetwo defining lesions in Alzheimer’s disease (AD) brain, theother being intracellular aggregation of hyperphosphory-lated tau into neurofibrillary tangles. Recent results indicatethat amyloid deposition begins ~10-20 years before theonset of dementia, suggesting that cerebral accumulation ofAβ has as a critical early role in AD pathogenesis [1-3]. Ifso, then inhibition of Aβ accumulation in the brain maybenefit AD, if given early enough during the course of thedisease.Neurons are the major producers of Aβ in the brain,

although glia, in particular astrocytes, may also contributeto Aβ generation, particularly during physiological stressthat causes glial activation as happens in AD. The forma-tion of Aβ is a sequential proteolytic process beginning withthe cleavage of amyloid precursor protein (APP) by the β-secretase enzyme, which generates the amino (N) terminus

Correspondence: r-vassar@northwestern.eduDepartment of Cell and Molecular Biology, Northwestern University, TheFeinberg School of Medicine, 303 E. Chicago Avenue, Chicago, IL 60611, USA

© 2014 Vassar; licensee BioMed Central. The licfollowing its publication. After this time, theLicense (http://creativecommons.org/licensemedium, provided the original work is propecreativecommons.org/publicdomain/zero/1.0

of Aβ and yields the membrane bound C-terminal fragmentC99 (Figure 1A) [4]. Next, γ-secretase cuts C99 to releaseAβ, which is secreted from the cell [5-7]. Interestingly, theγ-secretase cut is imprecise and creates Aβ isoforms ofdifferent lengths at the carboxy (C) terminus, of which thelonger isoforms are highly associated with AD. Processingof APP by both β- and γ-secretases is necessary for thegeneration of Aβ, suggesting that inhibition or modulationof either or both of these proteases in the brain shoulddecrease Aβ levels and be beneficial for AD.Human genetics studies have greatly informed us about

AD pathogenesis and strongly suggest that cerebral Aβaccumulation has an essential role in the etiology of AD[2]. Thus far, over 200 autosomal dominant mis-sensemutations have been identified in the genes for APP andpresenilin (the γ-secretase catalytic subunit) that are associ-ated with familial AD (FAD). These FAD mutations arehighly penetrant and without exception increase either thegeneration of all Aβ isoforms (total Aβ) or the relativeproportion of the 42-amino acid isoform (Aβ42) that ismore neurotoxic. Notably, FAD mutations in APP arefound very near to the β- and γ-secretase cleavage sites,and these mutations serve to increase APP processing andraise levels of total Aβ or Aβ42 specifically. The so-called

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Figure 1 APP processing and Aβ generation and mutations that affect β-secretase cleavage. A. APP is a Type-I membrane protein that issequentially cleaved by two aspartic proteases to generate Aβ. First, the β-secretase enzyme cuts APP (1) to create the N-terminus of Aβ. TwoAPP fragments are produced: membrane-bound C99 and secreted sAPPβ ectodomain. Second, C99 is cleaved by the γ-secretase enzyme (2) togenerate the C-terminus of Aβ. Aβ is then released into the lumen of the endosome and secreted into the extracellular medium. An intracellulardomain, C59, is also produced. B. The amino acids in and around the Aβ domain of APP are represented as blue circles. Amino acids that affectβ-secretase processing of APP in humans are green circles, within which the wild-type residue is identified by the single-letter amino acid code.The K670N/M671L (Swedish) and A673V mutations cause FAD by increasing β-secretase cleavage and Aβ production, while the A673T mutationprotects against AD by doing the opposite. All three mutations occur at or within one amino acid of the β-secretase cleavage site. Scissorsindicate cleavage sites of the various secretases.

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Swedish mutation (K670N; M671L) [8] and A673V [9]mutations in APP are particularly compelling, because theyare positioned precisely at and only 2 amino acids C-terminal to the β-secretase cleavage site, respectively. Thesemutations make the cleavage of APP by the β-secretaseenzyme more efficient, so greater amounts of C99 and totalAβ are generated (Figure 1B). In contrast, an APP muta-tion, A673T, has been recently identified that confersprotection against AD and cognitive decline in the elderly[10]. This mutation, which occurs at the same position asthe A673V mutation that causes FAD, is less efficientlycleaved by β-secretase so that Aβ generation is decreasedby ~40% [10-12]. Interestingly, most carriers have one copyof the A673T mutation and likely have a reduction in Aβproduction of only ~20%, yet they are still protected againstAD. This implies proof-of-principle of the strategy thatmodest reduction of brain Aβ levels may prevent AD, ifstarted early enough. Additionally, the Swedish, A673V, andA673T mutations together strongly suggest that inhibitionof β-secretase cleavage of APP should be beneficial for AD.

ReviewThe identification of β-secretase as β-site APP cleavingenzyme (BACE)Following the discoveries of Aβ and the first APP mutationsthat cause FAD, it soon became clear that the β- and γ-secretase enzymes were prime therapeutic targets for thedevelopment of small molecule inhibitor drugs for the

treatment of AD. Thus, their molecular identities werevigorously pursued. The properties of Aβ generation andsecretase activities in cells and tissues led to the develop-ment of cell-free and cell-based assays that could beexploited for the identification of the secretases. Subse-quently, five groups independently reported the molecularcloning of the β-secretase enzyme, which they variouslynamed β-site APP cleaving enzyme (BACE), Asp2, andmemapsin 2 [13-17] (“BACE” has emerged as the mostcommon moniker in the literature). Importantly, all of thegroups agreed on the same polypeptide sequence eventhough they used different experimental approaches toidentify the β-secretase, lending strong support for theconclusion that the authentic β-secretase had been cloned.BACE has all the molecular and cellular characteristics

that had been previously predicted for the β-secretasein vitro and in vivo [4]. It is a Type I transmembraneaspartic protease of 501 amino acids in length that is closelyrelated to the pepsin family of aspartic proteases (Figure 2).The catalytic domain of BACE harbors two aspartic prote-ase signature motifs of the sequence DTGS and DSGT thatcome together to form the active site of the enzyme. Asrequired for β-secretase, the BACE active site is topologic-ally oriented on the same side of the membrane as theβ-secretase cleavage site in APP. Additionally, the activityof BACE has an acidic pH optimum and the catalyticdomain resides within the lumen of acidic intracellular com-partments, including endosomes and trans-golgi network

Figure 2 Primary structure of BACE1. BACE1 is a 501 amino acidType-I transmembrane aspartic protease. The various subdomains ofBACE1 are indicated by the lines to the right of the structure. Numbersrefer to amino acid positions. The two signature aspartic proteaseactive site motifs at positions 93 and 289 are shaded yellow. S–Sdenote positions of disulfide bridges within the catalytic domain; Nsrepresents positions of N-linked glycosylation sites; Rs indicatespositions of acetylated arginine residues; Cs mark positions of S-palmitoylated cysteine residues; P indicates phosphorylation of serine498; Ub denotes ubiquitination of lysine 501.

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(TGN). Moreover, BACE levels are highest in neurons ofthe CNS, BACE has the correct sequence specificity, andBACE overexpressed in cells cleaves APP and increasesAβ production.

Soon after the discovery of BACE, a homologue, BACE2,was identified that has ~64% amino acid similarity to BACE(henceforth referred to as BACE1) [18]. The extensivedegree of homology between the two enzymes suggestedthat BACE2 might also function as a β-secretase. However,this possibility seemed unlikely because BACE2 is notexpressed to a high level in neurons, in contrast to BACE1[19,20]. Moreover, BACE2 predominantly cleaves APPwithin the Aβ domain, so that the generation of Aβ isprecluded [21-25]. These data, together with the findingthat BACE1 null mice are devoid of Aβ (see below), suggestBACE2 is not likely to be a β-secretase in the CNS.

Physiological functions of BACE1BACE1−/− miceTo justify BACE1 inhibitor drug development efforts, itwas necessary to provide in vivo validation that BACE1 isthe primary β-secretase enzyme in the brain. To do so, genetargeting in embryonic stem cells was used to produceBACE1 knockout (−/−) mice [26-29]. Initial reports showedthat BACE1−/− mice were viable and fertile and did nothave detectable abnormalities. Their normal morphologyand behavior, tissue histology, and blood cell and clinicalchemistry characteristics suggested that BACE1 inhibitionas a therapeutic approach for AD might lack mechanism-based toxicities. Additionally, APP overexpressing trans-genic mice that also lack the BACE1 gene are devoid ofcerebral Aβ, amyloid deposition, and Aβ-associated mem-ory impairments [20,30-33]. Importantly, these data validateBACE1 as the major β-secretase in the CNS and indicatethat BACE2 does not compensate for BACE1 loss offunction, at least for the production of Aβ. Furthermore,they strongly suggested that BACE1 inhibition should be asafe and effective therapeutic strategy for AD.Although initial studies of BACE1−/− mice indicated that

BACE1 was not required for viability in vivo, further inves-tigations were necessary to elucidate the physiologicalfunctions of BACE1 and fully understand the potential formechanism-based toxicities of therapeutic BACE1 inhib-ition. For example, BACE1 protein is highly concentratedin presynaptic terminals of CNS neurons [34,35], suggestingthat BACE1 has a role in synaptic function. Moreover, inagreement with high BACE1 expression and presynapticlocalization in neurons, deeper analyses of BACE1−/− micehave uncovered numerous subtle neuronal phenotypes,such as axon targeting errors [36-38], reduced myelination[39-41], memory impairments [20,30,32,42,43], reducedmuscle spindles [44], neurochemical abnormalities [45],alterations in neurogenesis and astrogenesis [46], increasedage-related neurodegeneration [47], reduced spine density[48], retinal pathology [49], endophenotypes of schizophre-nia [48], and seizures [42,47,50] (Table 1). Future investiga-tions may reveal even more BACE1 null phenotypes. Anyof these BACE1 null phenotypes in theory could represent

Table 1 BACE1 knockout mouse phenotypes

Phenotype PutativeSubstrate

References

Astrogenesis increase; neurogenesisdecrease

Jag1 [46]

Axon guidance defects CHL1 [36-38]

Hyperactivity NRG1 [29,48]

Hypomyelination NRG1 [39-41]

Memory deficits Unknown [17,30,32,42,43]

Insulin sensitivity enhanced Unknown [29,51,52]

Muscle spindle reduction NRG1 [44]

Neurochemical deficits Unknown [45]

Neurodegeneration with age Unknown [47]

Postnatal lethality, growth retardation Unknown [29]

Retinal abnormalities VEGFR1 [49]

Schizophrenia endophenotypes NRG1 [48]

Seizures Navβ2 [42,47,50,53]

Spine density reduction NRG1 [48]

BACE2 knockout mouse phenotypes

Phenotype PutativeSubstrate

References

Normal — [29]

Pancreatic β cell increase Tmem27 [54]

Pigmentation abnormalities PMEL [55]

BACE1/2 double knockout mouse phenotypes

Phenotype References

Similar to BACE1 knockout, except postnatal lethalityis enhanced

[29]

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mechanism-based side effects of BACE1 inhibitor drugs inhumans, thus raising a note of caution that therapeutic in-hibition of BACE1 might not be completely free of toxicity.

Substrates of BACE1The varied phenotypes of the BACE1−/− mice are likelythe result of abrogated β-secretase processing of differentsubstrates of BACE1 in addition to APP. Recent proteomicanalyses in cultured primary neurons have identified nu-merous putative BACE1 substrates that have roles in neur-onal functions [56,57] (Figure 3). The majority of substratesof BACE1 are, like APP, Type I membrane proteins, while afew, like neuregulin 1 (NRG1), have more complex mem-brane topologies. Cleavage of most substrates by BACE1releases an ectodomain fragment that diffuses from the cellin the extracellular milieu. There, it may bind to anothermolecule on the same (autocrine) or a different (paracrine)cell to affect signal transduction or cell-cell interactions.Perhaps the best studied example is that of BACE1 process-ing of Type III NRG1, which releases an epidermal growthfactor (EGF)-like domain that binds to the ErbB receptoron the Schwann cell for the simulation of myelination

[39,40,58,59]. Because of the lack of β-secretase processing,BACE1−/− mice have decreased shedding of the NRG1EGF domain, which reduces instructive signals to myelinat-ing cells and leads to hypomyelination.Another example of β-secretase processing of a neuronal

substrate involves the cleavage of the neural cell adhesionmolecule close homolog of L1 (CHL1) by BACE1. LikeAPP, CHL1 is a Type I membrane protein, and it has awell-known function in axonal outgrowth and neuronalsurvival [60,61]. Cleavage of CHL1 by BACE1 liberates asoluble ectodomain fragment that may bind to neuropilin-1and semaphorin 3A, two molecules that are involved inaxonal guidance. Thus, the lack of β-secretase processing ofCHL1 might account for the presence of mis-targetedaxons that have been reported in the olfactory bulb andhippocampus of BACE1 null mice [38,56,57].Although decreased β-secretase processing of many

BACE1 substrates impairs their function, abrogated cleav-age of other substrates may potentiate their role in aphysiological process. For example, Jagged 1 (Jag1) is aType I membrane protein that is a ligand for the Notch re-ceptor, which regulates the differentiation of many celltypes in the body. Interestingly, Jag1 is also a BACE1 sub-strate, and reduced BACE1 cleavage of Jag1 in BACE1−/−mice increases the levels of Jag1 on the cell surface, whichcauses greater than normal stimulation of Notch activity inthe neighboring cell. Consequently, during early develop-ment Jag1-Notch signaling is increased [62] in radial glialneural stem cells, which promotes astrogenesis overneurogenesis [46]. As additional BACE1 substrates andfunctions are discovered, the underlying molecular mecha-nisms of BACE1 null phenotypes and their implications formechanism-based toxicities of therapeutic BACE1 inhib-ition will come into clearer focus.In addition to cleavage by BACE1, a number of BACE1

substrates undergo ectodomain shedding by proteases inthe A Disintegrin and Metalloproteinase Domain (ADAM)family. The extent to which a given substrate is processedby BACE1 verses an ADAM family member varies depend-ing on the substrate (Figure 3). Some substrates are almostexclusively cut by BACE1 (e.g., SEZ6, APLP1), while othersubstrates are primarily cleaved by the ADAMs (e.g., APP,neuroligin-1) [56,57]. One would predict from these resultsthat potential mechanism-based side effects that arise fromtherapeutic BACE1 inhibition might derive from deficientprocessing of substrates that predominantly undergo ecto-domain shedding by BACE1 rather than the ADAMs.Conversely, potential toxicities of BACE1 inhibition may beless associated with substrates that are primarily cleaved byADAM proteases over BACE1.

BACE2−/− miceThe significant amino acid similarity shared by BACE1 andBACE2 suggests that it may be challenging to develop

Figure 3 Neuronal substrates of BACE1. BACE1 substrates identified in primary cultured neurons are listed from those that are predominantlycleaved by BACE1 (BACE1 cleavage HIGH; top) to those that are processed by BACE1 at a low level (LOW; bottom). These substrates also arecleaved by other proteases in the ADAM family, but the ADAM cleavage preference is opposite to that of BACE1. (Adapted from Table I, Ref. [56]).

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BACE1 inhibitors that do not cross-inhibit BACE2. There-fore, the possibility exists that BACE1 inhibitor drugs mightalso cause BACE2 mechanism-based side effects in additionto those of BACE1. To investigate this question, BACE2−/−mice were produced by gene-targeting. Like BACE1 nullmice, the BACE2−/− mice were initially shown to be viableand fertile with no reported phenotype [29]. Moreover,other than enhanced early postnatal lethality, BACE1−/−;BACE2−/− double knockout mice did not have a moresevere phenotype than the BACE1−/− single knockouts[29]. These data suggest that cross-inhibition of BACE2with BACE1 inhibitors might not be associated withenhanced toxicity in the adult after postnatal developmentis completed.Although BACE2−/− mice initially were reported to be

normal, further investigations have revealed BACE2 loss-of-

function phenotypes. Pancreatic β-cells express significantlevels of BACE2. Interestingly, BACE2−/− mice haveincreased β-cell mass and insulin levels, and the mice ex-hibit enhanced glucose regulation [54]. These phenotypesappear to be the result of abrogated BACE2 cleavage ofpro-proliferative Type I transmembrane protein Tmem27,a protein involved in the regulation of β-cell mass. Giventhese results, inhibition of BACE2 may be beneficial for thetreatment of Type 2 diabetes, although further researchinto this hypothesis is necessary.In addition to the pancreatic phenotype, BACE2−/− mice

on a C57BL/6 genetic background exhibit hypopigmentationthat results in a silvery coat compared to the dark coat ofwild-type C57BL/6 mice. This phenotype is caused by lackof BACE2 processing of the melanocyte protein PMEL thatis expressed in pigment cells of the skin and eye. BACE2

Table 2 Small molecule BACE1 inhibitors in clinical trials

Company Drug Phase

AstraZeneca/Lilly AZD3293 Phase 2/3

CoMentis CTS-21166 Phase 1

Eisai/Biogen Idec E2609 Phase 2

High Point HPP854 Phase 1

Janssen/Shionogi - - - - - - Phase 1

Lilly LY2886721 Phase 2*

Merck MK-8931 Phase 2/3

Novartis - - - - - - Phase 1

Pfizer PF-05297909 Phase 1

Roche RG7129 Phase 1**

Takeda TAK-070 Phase 1

Vitae/Boehringer Ingelheim VTP-37948 Phase 1

*Terminated due to abnormal liver biochemistry.**Removed from pipeline.

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cleavage releases a fragment of PMEL into melanosomesthat forms a matrix of amyloid fibrils upon which melaninis deposited [55]. Consequently, abrogated processing ofPMEL in BACE2−/− mice leads to abnormal melanosomeformation and hypopigmentation. These results suggest thepossibility that cross-inhibition of BACE2 by BACE1 inhibi-tors might cause reduced pigmentation in humans.

Small molecule BACE1 inhibitor drugs and clinical trialsfor ADThe extensive validation of BACE1 as the primary β-secretase enzyme in the CNS has spurred vigorous effortsto develop small molecule inhibitors of BACE1 in both aca-demia and industry. The first generation of BACE1 inhibi-tors consisted of non-cleavable peptide-based transitionstate analogs designed after the amino-acid sequence inAPP at which β-secretase cleaves [15,63]. Typically, theselarge peptidomimetic molecules are very potent BACE1inhibitors in vitro, mainly because the large open active siteof BACE1 has evolved to bind polypeptide substrates withhigh affinity. Unfortunately, the peptide-based BACE1 in-hibitors did not possess favorable in vivo pharmacologicalproperties, such as oral bioavailability, long serum half-life,or blood–brain barrier (BBB) penetration. As a conse-quence, investigators have turned toward designing truesmall molecule BACE1 inhibitor drugs. However, the devel-opment of non-peptidic BACE1 inhibitors large enough tobind with sufficient affinity to the enzymatic active site, yetsmall enough to exhibit satisfactory pharmacokinetics andsuitable brain penetration has proven to be very challen-ging. Moreover, BACE1 inhibitors should have sufficientlipophilicity to cross both plasma and endosomal mem-branes for gaining access to the vesicle lumen where theBACE1 active site is located.A crucial advance in small molecule BACE1 inhibitor de-

velopment came with the first X-ray co-crystal structure ofBACE1 with a peptidic BACE1 inhibitor [64]. The BACE1X-ray structure revealed important inhibitor-enzyme inter-actions that were exploited in rational drug design efforts.Shortly thereafter, new classes of small molecule BACE1 in-hibitors were developed that exhibited improved pharma-cological characteristics, including small molecular weight,plasma membrane permeability, and better pharmacokinet-ics [65,66]. However, most second-generation BACE1inhibitors were substrates of P-glycoprotein, the ATP-dependent drug efflux pump for xenobiotics in the BBB[67], and therefore could not reach high concentrations inthe brain.More recently, potent third-generation small molecule

BACE1 inhibitors have been developed that achieve satis-factory brain penetration and robust cerebral Aβ reductionin preclinical animal models. Innovative diverse and com-plex drug development approaches have been employed todesign current BACE1 inhibitors, which are described in

detail in recent reviews [65,66]. Several of these orallybioavailable BACE1 inhibitor drugs have entered intohuman clinical trials (Table 2). Most are in the early clinicalphases and scant information on their progress has beenpublished, although preliminary trial results for threeBACE1 inhibitor drugs have been reported at recentconferences and are summarized below.

LY2886721The pharmaceutical company Eli Lilly was among the firstto develop and test orally bioavailable non-peptidic BACE1inhibitors in humans. The small molecule BACE1 inhibitorLY2811376 showed satisfactory pharmacokinetic and phar-macodynamic characteristics in preclinical animal modelsthat translated to a Phase 1 clinical trial in humans [68].However, chronic toxicology studies in rat showing non-clinical non-target associated pathology in retina and brainprecluded the clinical development of this molecule.Although it was discontinued, LY2811376 demonstrated thefeasibility of developing a potent brain-penetrant orally bio-available small molecule BACE1 inhibitor and representedthe first reported translation of reduced CSF biomarkers ofBACE1 cleavage from preclinical animal models to humans.Lilly advanced a next-generation compound, LY2886721,

into Phase 1 and 2 clinical trials to determine its safety andtolerability, pharmacokinetics, and pharmacodynamics.Similar to LY2811376, LY2886721 was a potent orallybioavailable small molecule BACE1 inhibitor that robustlydecreased levels of Aβ in the brains of preclinical animalmodels. However, unlike LY2811376, treatment withLY2886721 did not appear to be toxic to the retina or brain.Forty-seven healthy volunteers were given daily oral dosesof either LY2886721 or placebo for 14 days in Phase 1 [69].Either a multiple ascending dose (5, 15, and 35 mg) or asingle dose (70 mg) followed by a multiple ascending

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dose was performed in two Phase 1 study designs(NCT01227252, NCT01534273). Over the course of the14-day study, LY2886721 was reported to be safe and welltolerated. Plasma half-life of LY2886721 was ~12 hours,compatible with once per day dosing. Dose-dependent de-creases of both plasma and CSF Aβ40 levels resulted fromLY2886721 administration. Aβ40 levels in the CSF were de-creased up to 74% with the highest dose of LY2886721.Levels of Aβ42 and sAPPβ in the CSF were both reducedto a similar extent as CSF Aβ40 by LY2886721. Interest-ingly, levels of sAPPα, the α-secretase cleavage product,were increased in CSF [70], an observation that is consist-ent with BACE1 inhibition, since β- and α-secretase com-pete for processing of APP. A Phase 1 study of LY2886721in patients with AD was also conducted (NCT01807026).The positive Phase 1 trials led to a six-month Phase 2

trial of 35 or 70 mg of LY2886721 dosed orally once perday in 130 patients with prodromal AD, also known asamnestic mild cognitive impairment (MCI), or mild AD(NCT01561430) [71]. Recently, Lilly voluntarily terminatedthe Phase 2 trial because a small number of subjects thatwere given LY2886721 developed abnormal liver biochem-istries. The company reported that the LY2886721-relatedliver abnormalities did not appear to be associated with theBACE1 mechanism of action, a conclusion supported by anormal liver phenotype of BACE1−/− mice. It is notuncommon that some small molecules in clinical develop-ment are discontinued because of abnormal liver functionas a non-target related side effect. Thus, the termination ofLY2886721 does not necessarily suggest that BACE1 is nota viable drug target.

MK-8931MK-8931, a small molecule BACE1 inhibitor developed bythe pharmaceutical company Merck was tested in 88healthy volunteers (18–45 years old) as a two-part random-ized, double-blind, placebo-controlled Phase 1 clinical trial[72]. Single and multiple (daily for 14 days) oral doses ofMK-8931 were analyzed for safety, tolerability, pharmaco-kinetics, and pharmacodynamics. In healthy volunteers,MK-8931 was well tolerated and no serious adverse eventswere reported. Determining whether MK-8931 was able toenter the brain and engage its target, the β-secretaseenzyme, were primary goals of the study. To do so,biomarkers of BACE1 activity were measured in the CSF,including Aβ40, Aβ42, and sAPPβ, the latter being theBACE1-cleaved ectodomain of APP. MK-8931 markedlyreduced levels of Aβ in the CSF in a sustained and dose-dependent manner. A single oral dose of 100 or 550 mg ofMK-8931 decreased CSF Aβ40 levels by 75% or 92%,respectively, at 36 hours after dosing. Levels of Aβ42 andsAPPβ in the CSF were also reduced to similar extents.Multiple oral dosing of MK-8931 lowered Aβ levels in theCSF by over 90%. MK-8931 has a plasma half-life of

~20 hours, suggesting that a single daily oral dose maymaintain stable drug levels in vivo.A randomized, double-blind, placebo-controlled Phase

1b trial of MK-8931 in 32 mild to moderate AD patients(mean age and Mini-Mental State Examination (MMSE),73 yrs. and 22, respectively) was also conducted for safety,tolerability, pharmacokinetics, and pharmacodynamics(NCT01496170) [73]. One of three doses (12, 40, or60 mg) of MK-8931 or placebo was given once each dayorally for 7 days and levels of Aβ40, Aβ42, and sAPPβ inthe CSF were measured. Similar to the healthy volunteers,MK-8931 strongly decreased levels of Aβ in the CSF in asustained and dose-dependent fashion. Daily dosing of 12,40, or 60 mg reduced CSF Aβ40 by 57, 79, or 84%,respectively, and resulted in similar reductions for CSFAβ42 and sAPPβ. MK-8931 did not appear to cause ser-ious adverse events in the AD patients during the courseof the study. Importantly, the MK-8931 Phase 1b resultssuggest that the pharmacokinetic and pharmacodynamicproperties of BACE1 inhibitor drugs are not significantlyaltered by the presence of high amyloid loads in the brainsof AD patients.Encouraged by the positive results of the MK-8931 Phase

1 and 1b studies, a Phase 2/3 combined clinical trial (theEPOCH study, NCT01739348) was started in late 2012.EPOCH is a 78-week, randomized, placebo-controlled,parallel-group, double-blind clinical trial to evaluate thesafety and efficacy of 12 or 40 mg/day oral dosing of MK-8931 versus placebo in mild to moderate AD patients. InPhase 2 the trial will evaluate 200 AD patients and willenroll up to 1,700 patients for Phase 3. Primary efficacyoutcomes are the changes from baseline in the Alzheimer’sDisease Assessment Scale Cognitive Subscale (ADAS-Cog)and the Alzheimer’s Disease Cooperative Study-Activitiesof Daily Living (ADCS-ADL) scores.A recent interim safety analysis in 200 AD patients

treated with MK-8931 for at least 3 months suggested thatthe drug was well tolerated and that the EPOCH studyproceed without changes to the protocol. Enrolment in thetrial has continued with up to 1960 patients expected forPhase 3. An additional clinical trial (the APECS study,NCT01953601) has also commenced, consisting of a104 week randomized, placebo-controlled, parallel-group,double-blind Phase 3 study to evaluate the safety andefficacy of 12 mg or 40 mg per day oral dosing of MK-8931versus placebo in 1500 patients with MCI. The primaryefficacy outcome in APECS is the change from baseline inthe Clinical Dementia Rating Scale-Sum of Boxes (CDR-SB) score. Secondary outcome substudies are included inboth EPOCH and APECS to measure AD biomarkers,consisting of cortical amyloid load, CSF Aβ and tau, andhippocampal volume. The Phase 3 efficacy studies forEPOCH and APECS are expected to conclude in 2017 and2018, respectively.

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AZD3293The AstraZenica BACE1 inhibitor, AZD3293, was recentlytested for safety, tolerability, pharmacokinetics, and effectson plasma and CSF Aβ levels in healthy young (18–55 yr)and elderly (55–80 yr) subjects [74]. Phase 1 randomized,double-blind, placebo-controlled SAD and MAD studieswere conducted. In the SAD study (NCT01739647), 1 to750 mg doses of AZD3293 were administered to 7 youngcohorts, while an elderly cohort received 15 mg (8 subjects/cohort). In the MAD study (NCT01795339), multipleonce-daily doses of AZD3293 ranging from 15 to 70 mgwere administered to 5 cohorts (two elderly) for 2 weeks.AZD3293 was well tolerated with no serious adverse eventsreported up to the highest dose given (750 mg) in the SADstudy. The half-life of AZD3293 was 11–20 hours and thuscompatible with once daily dosing. Pharmacokinetic param-eters of AZD3293 between elderly and young subjects wereindistinguishable. In the MAD study, the 15 or 50 mg dosesreduced CSF Aβ40 and Aβ42 concentrations by a constant50 or 75%, respectively. Additionally, AZD3293 administra-tion produced dose-dependent decreases and increases ofsAPPβ and sAPPα concentrations in the CSF, respectively,that had similar timelines as the reductions in CSF Aβ40and Aβ42 [75]. The Phase 1 studies of AZD3293 in healthsubjects (NCT01739647) and AD patients (NCT01795339)have been completed, and combined Phase 2/3 trials in1,551 MCI and mild AD patients are planned (20 mgor 50 mg doses, 104 week duration, AMARANTH trial(NCT02245737)). Recently, AstraZenica and Lilly enteredinto a partnership to jointly develop AZD3293 for AD.

E2609The drug company Eisai has developed an orally bio-available small molecule BACE1 inhibitor, E2609, that hasshown robust lowering of cerebral Aβ in preclinical andclinical studies. E2609 was first clinically studied inhealthy volunteers in randomized, double-blind, placebo-controlled Phase 1 trials [76-78]. A single oral ascendingdose (SAD) study (73 subjects) and a 14-day multiple oralascending dose (MAD) study (50 subjects) tested E2609 intwo separate Phase 1 clinical trials (NCT01294540 andNCT01511783, respectively). The SAD study analyzedplasma Aβ levels following E2609 administration rangingfrom 5 to 800 mg (9 cohorts), while the MAD studymeasured both plasma and CSF Aβ levels in response toE2609 doses ranging from 25 to 400 mg (5 cohorts). TheE2609 plasma half-life of 12–16 hours is compatible withonce per day dosing. Each of the two Phase 1 studiesshowed robust dose-dependent decreases of levels of Aβin the CSF and/or plasma. CSF Aβ levels were reduced upto 85% at the highest dose of E2609 (400 mg) in the MADstudy. Similar decreases in levels of sAPPβ in the CSFwere observed, while CSF levels of sAPPα were increased.E2609 appeared to be safe and well tolerated, as no serious

adverse events were reported in either Phase 1 study. Eisaihas recently completed a Phase 1 trial of E2609 in subjectswith MCI or mild AD (NCT01600859), and a Phase 2clinical trial of E2609 is planned. Recently, Eisai andBiogen Idec entered into a partnership to jointly developE2609 for AD.

Alternative therapeutic approaches for BACE1 inhibitionAlthough small molecules that directly inhibit BACE1 en-zyme activity are leading therapeutic approaches, potentialalternative strategies to reduce BACE1 processing of APPare being explored. As noted, BACE1 levels are significantlyelevated in AD brain and might accelerate the productionof Aβ. Therefore, approaches to lower and normalizeBACE1 levels in the brain might slow AD progression andavoid possible untoward side effects caused by directBACE1 enzyme inhibition. Consequently, efforts are under-way to elucidate the mechanisms of BACE1 elevation inAD in order to identify drug targets that could block theBACE1 increase when inhibited. BACE1 undergoescomplex regulation at the transcriptional, translational, andpost-translational levels, all of which appear to have a rolein elevating BACE1 levels and activity in AD [79-81]. Muchevidence suggests that BACE1 is a stress response proteasethat is increased by oxidative stress, inflammation, hypoxia,and trauma, among other insults that occur in AD[79,82,83]. Even Aβ itself increases BACE1 levels in neu-rons [84,85], suggesting a vicious pathogenic cycle wherebyAβ could accelerate its own production through BACE1elevation. Which, if any, of these complex multi-layeredregulatory mechanisms might yield therapeutic strategiesfor lowering BACE1 levels in AD is unclear, but continuingresearch in this important area may reveal promising newAD drug targets in the future.Another class of alternative therapeutic strategy for

BACE1 inhibition involves immunotherapy approaches toreduce BACE1 processing of APP. The first of these strat-egies employs antibodies directed against the β-secretasecleavage site of APP that sterically block access of theBACE1 active site to APP [86,87]. These anti-β-site APPantibodies decrease Aβ production cultures cells and wheninjected i.v. reduce amyloid plaque pathology in the brainsof APP transgenic mice [88]. Other immunotherapyapproaches include anti-BACE1 antibodies that are notdirected against the active site but instead target an exositeon the surface of the BACE1 catalytic domain that canallosterically regulate enzyme activity [89,90]. This exositeis located on structurally adjacent regions of the C, D, andF loops of the enzyme [91]. Exosite antibody binding toBACE1 alters structural features and dynamic characteris-tics near the substrate binging cleft of the enzyme.Additionally, transport of BACE1 antibodies across theBBB has been facilitated by engineering one arm of theantibody to recognize transferrin receptor (TfR), which

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shuttles transferrin across the BBB for the delivery of ironinto the brain [92,93]. These bispecific BACE1-TfR anti-bodies accumulate in the brain and reduce endogenous Aβlevels in mice to a much greater extent than monospecificBACE1 antibodies. Moreover, TfR bispecific antibodiescould be useful for treating other neurologic diseasesamenable to immunotherapy. These antibody approachesare currently in preclinical phases.

Unanswered questions that are relevant to BACE1 inhibitorclinical trialsFifteen years after the discovery of the β-secretase enzyme,the challenges of developing brain-penetrant BACE1 inhibi-tors have been accomplished and human clinical trials areunderway. This promising development raises hopes thatdisease-modifying therapies employing BACE1 inhibitionfor AD are within reach. However, important questionsconcerning therapeutic goals and outcomes of these trialsremain to be answered:

What degree of BACE1 inhibition will be needed to achieveefficacy?The level of BACE1 inhibition required for efficacy in turnshould depend on how much Aβ lowering is necessary andat what stage of AD to treat (questions discussed furtherbelow). The recently discovered A673T APP mutation thatprotects against AD [10] suggests that reducing cerebral Aβproduction by only a modest amount (~20%) could be pre-ventative, if started before significant amyloid accumulation.As discussed above, the leading BACE1 inhibitors currentlyin clinical trial are capable of this relatively small Aβdecrease. How BACE1 inhibition translates to Aβ reductionin the brain is difficult to estimate, although some insightinto this question may be gained by considering experi-ments in BACE1 knockout mice. Heterozygous BACE1+/−mice that model 50% therapeutic inhibition of BACE1exhibit ~20% lowering of cerebral Aβ levels in APP trans-genic mice [20,33]. Importantly, BACE1+/− mice appear tobe normal, so 50% BACE1 inhibition may circumventmechanism-based side effects yet provide sufficient Aβreduction for efficacy.As suggested by the protective A673T mutation, a thera-

peutic approach that reduced BACE1 activity and Aβ levelsby ~50% and ~20%, respectively, would probably need tostart before major amyloid deposition and be maintainedfor the remainder of life to prevent or delay the onset ofAD. However, inhibiting BACE1 by more than 50% couldbe required if significant amyloid plaque load is present inthe brain at the beginning of treatment. Still, the possibilityexists that no level of BACE1 inhibition, no mater howstrong, would be able to slow the progression of AD once acertain threshold of amyloid burden is reached. At present,these arguments are all speculative, as the levels of BACE1inhibition and Aβ reduction necessary for efficacy in

humans are as yet unknown, although insight into theseparameters might be gleaned following analysis of theresults from the ongoing clinical trials.It is important to note that cerebral BACE1 levels in AD

patients are increased by several fold over those in normalindividuals [94-97]. Both BACE1 and APP accumulate inswollen dystrophic neurites that surround amyloid plaques[34,98,99], suggesting increased peri-plaque Aβ productionthat might accelerate amyloid deposition and induce avicious pathogenic cycle [100]. If so, normalization ofBACE1 activity in peri-plaque dystrophic neurites mayrepresent a modest but potentially efficacious therapeuticgoal of BACE1 inhibition. However, elevated concentrationsof BACE1 around plaques might necessitate the administra-tion of very high BACE1 inhibitor doses in order to signifi-cantly reduce peri-plaque Aβ generation, if the amyloidburden is great.

What stage of AD should we administer BACE1 inhibitors?Cerebral Aβ accumulation has a crucial early role in ADpathogenesis, as suggested by over 200 FAD mutations [2].Amyloid deposition appears to begin more than a decadebefore the manifestation of cognitive deficits and theclinical diagnosis of AD [101-103]. Aβ-lowering BACE1inhibitors are likely to be most effective as a preventionstrategy when administered early in the course of AD,before significant cerebral amyloid accumulation andneurodegeneration. Thus, BACE1 inhibitors are analogousto the cholesterol-lowering statin drugs for the preventionof heart disease: once significant amounts of cholesterolhave deposited in coronary arteries and major injury to theheart has occurred, statin administration is unable to re-verse the damage and provide much benefit for the patient.AD prevention trials will necessarily involve the enrollmentof thousands of subjects, last for years, and incur enormouscosts. As a result, AD prevention trials might be mostfeasible in the context of joint government-industry collab-orations, such as those being conducted or planned by theAnti-Amyloid Treatment in Asymptomatic Alzheimer’sDisease (A4) trial, Alzheimer’s Prevention Initiative (API),and Dominantly Inherited Alzheimer Network Trials Unit(DIAN TU). Some AD prevention trials are enrollingcognitively normal individuals that are genetically at highrisk for developing AD who carry autosomal dominantFAD mutations (DIAN TU trial) or are homozygous forthe apolipoprotein E ε4 allele (API trial). If BACE inhibitorsare as well tolerated in chronic dosing for AD as the statinsare for lowering serum cholesterol to prevent heart disease,then treating pre-symptomatic at-risk individuals for ADprevention is warranted.Current BACE1 inhibitor trials have enrolled mild and

moderate AD or mild cognitive impairment (MCI), thelatter of which progresses to AD at a rate of ~10-15% peryear [104]. A major advance has been the development of

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amyloid positron emission tomography (PET) imaging andCSF Aβ42 measurement as biomarkers for the diagnosis ofprodromal AD [105,106]. Individuals that exhibit significantamyloid load by PET or have reduced CSF Aβ42 concentra-tions are likely to develop AD, even though they appearcognitively normal at the time of testing. Due to theunavailability of amyloid PET or CSF Aβ42 biomarker test-ing at the time, past Aβ immunotherapy trials were unableto exclude subjects that did not have cerebral amyloid accu-mulation, thus leading to increased variability in cognitiveoutcome results and ultimately contributing to the frankfailure of these clinical trials [107]. In contrast, the BACE1inhibitor clinical trials are enrolling only subjects that arepositive by amyloid PET or CSF Aβ42, which shoulddecrease data variability and increase the probability ofobserving statistically significant differences in cognitionbetween drug and placebo groups. Periodic amyloid PET orCSF Aβ42 testing will be conducted to monitor targetengagement and amyloid accumulation over the courses ofthe trials. Cognitive performance will also be tested, as thismeasure is the gold standard for efficacy in past AD clinicaltrials of approved palliative drugs that treat memory symp-toms. However, as noted above, amyloid deposition appearsto start years before memory deficits are detected withcurrent tests of cognition. Thus, it might be challenging forBACE1 inhibitors to alter the trajectory of AD once a largeamount of amyloid has accumulated in the brain, at leastregarding the reduction of cognitive decline.It is hoped that the levels of BACE1 inhibition and Aβ

reduction necessary for disease modification could bededuced from data collected at the conclusion of thecurrent clinical trials. Pharmacodynamic models developedfrom this future data might assist in the estimation of thelevel of BACE1 inhibition required to achieve efficaciousAβ reduction for a given cerebral amyloid load and level ofcognitive impairment. These models could also be usefulfor the design of future primary and secondary AD preven-tion trials in pre-symptomatic individuals. At this time, therelationships between BACE1 inhibition, Aβ reduction,amyloid load, and cognitive status are not sufficiently wellunderstood to develop accurate pharmacodynamic modelsfor estimating the levels of BACE1 inhibition needed at agiven stage of asymptomatic or symptomatic AD.

Will treatment with BACE1 inhibitors cause mechanism-basedside effects?Although BACE1−/− mice were initially reported to benormal, recent studies have identified over a dozen BACE1null phenotypes and substantially more BACE1 substrates(Table 1, Figure 3), suggesting therapeutic BACE1 inhib-ition might cause mechanism-based toxicities. That said, itis unclear to what extent BACE1 null phenotypes in miceare able to model potential BACE1 inhibitor side effects inhumans, for several reasons. First, BACE1 null phenotypes

could relate to functions of BACE1 either during develop-ment or in adulthood, since BACE1−/− mice lack BACE1from conception. For example, the major proportion ofmyelination occurs during development and is completedwhen adulthood is reached [108], indicating that hypomye-lination as a result of abrogated BACE1 processing ofNRG1 in BACE1−/− mice is a developmental phenotype.Consequently, BACE1 inhibition in the adult might nothave an impact on myelination, unless re-myelination fol-lowing injury becomes necessary. In contrast, neurogenesisand axon guidance are ongoing processes that occur inspecific neuronal subpopulations that regenerate through-out life [38,46], suggesting the possibility that BACE1−/−abnormalities in neurogenesis and axon targeting are adultphenotypes and that BACE1 inhibitor treatment might leadto similar defects. Additionally, it is possible that compen-sation from other proteases during development couldmitigate the effects of BACE1 null mutation, in which caseBACE1 inhibitor treatment in humans might have more se-vere side effects than indicated by BACE1−/− mice. Giventhese arguments, comprehensive analyses of BACE1−/−mice should help to parse developmental verses adultBACE1 null phenotypes for the estimation of BACE1inhibitor side effect risk.The risk of BACE1 mechanism-based toxicities will

depend in large part on the degree of therapeutic BACE1inhibition. At one extreme, BACE1−/− mice model 100%BACE1 inhibition, but this level of inhibition will never beachieved by BACE1 inhibitor treatment in practice, thusreducing the chance of side effects. However, AD patientsare elderly and often frail, thus increasing the risk of seriousadverse events caused by BACE1 inhibition. Moreover,BACE1 inhibitors must be chronically administered, neces-sitating a high level of safety. Ongoing and future BACE1inhibitor clinical trials will ultimately answer these ques-tions. It is anticipated that a therapeutic window will bediscovered in which an empirically determined range ofBACE1 inhibitor doses can balance tolerable mechanism-based side effects with sufficient reduction of cerebral Aβlevels for efficacy.The statins are useful as a group for modeling the clin-

ical development of BACE1 inhibitors, as indicated by thefact that the statin clinical trials determined a therapeuticdose window of HMG Co-A reductase inhibitor thateffectively decreased serum cholesterol levels to preventheart disease in the presence of tolerable side effects. Weare now in the early phases of this clinical developmentmodel for BACE1 inhibitors. Regardless of the final out-comes of the current BACE1 inhibitor trials, invaluableknowledge will be gained about the quantitative andtemporal relationships between BACE1 inhibition, Aβreduction, amyloid burden, and cognitive function inhumans, which will be used for future clinical developmentof BACE1 inhibitors for AD.

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ConclusionsAs the β-secretase enzyme that initiates the production ofAβ, BACE1 is a key therapeutic target for AD. The protect-ive A673T APP mutation in humans and genetic deletionof BACE1 in mice both decrease Aβ generation via reducedβ-secretase processing of APP, providing strong proof ofconcept that BACE1 inhibition should be efficacious forAD. However, BACE1 null mice exhibit multiple complexneurological phenotypes (Table 1), suggesting that BACE1inhibitor drugs might cause mechanism-based side effectsinvolving hypomyelination, seizure, axon guidance defects,memory deficits, neurogenesis abnormalities, and neuro-degeneration, and potentially others, resulting from insuffi-cient BACE1 processing of a growing list of BACE1substrates in neurons. Which, if any, of these BACE1 nullphenotypes model BACE1 inhibitor side effects in humansremains to ne determined.Despite the challenges of BACE1 inhibitor drug develop-

ment over the past 15 years since the discovery of BACE1,the recent introduction of several BACE1 inhibitors intoclinical trials has refocused attention on this promisingtherapeutic approach for AD. To date, Merck’s MK-8931has advanced the farthest to Phase 2/3, while the otherdrugs including those from AstraZeneca, Eisai, and Pfizer,among others, are in Phases 1 and 2. These compounds arepotent, achieving up to ~90% CSF Aβ reduction. Addition-ally, they are well-tolerated for the most part, although twoBACE1 inhibitors have recently been terminated due totoxicity that might not be related to the BACE1 mechanismof action.The most challenging questions for BACE1 inhibitor clin-

ical development concern the level of BACE1 inhibitionand the stage of AD at which to treat for optimal efficacy.Hypothetical arguments based on the A673TAPP mutationand BACE1+/− mice suggest that ~50% BACE1 inhibitionmight achieve ~20% Aβ reduction, which could preventAD if begun well before significant amyloid deposition.However, it is unclear whether any level of BACE1 inhib-ition can be effective if major amyloid accumulation ispresent in the brain. Amyloid PET imaging, CSF Aβ42measurement, and other biomarker studies suggest thatamyloid deposition starts years, even decades, before theclinical diagnosis of dementia. Moreover, the relationshipsbetween amyloid burden and cognitive impairment are notsufficiently well understood to determine the stage of ADthat BACE1 inhibitor treatment would be most effective.Results from ongoing biomarker studies, future treatmentand prevention trial, and pharmacodynamic modeling areexpected to establish the appropriate level of BACE1 inhib-ition and stage of AD for optimal efficacy. Like the statinsfor hypercholesterolemia, the hope is that a therapeuticwindow of BACE1 inhibitor doses might be found that re-duces cerebral Aβ levels enough for efficacy, yet maintainssufficient BACE1 activity for the avoidance of side effects.

When eventually published, the results of the currentBACE1 inhibitor clinical trials will prove invaluable forsolving these important questions. We are at a crucialjuncture in BACE1 inhibitor drug development, and thetherapeutic potential of BACE1 inhibition for AD will bedefinitively answered in the not too distant future.

AbbreviationsAD: Alzheimer’s disease; APP: Amyloid precursor protein; Aβ: β-amyloid peptide;BACE1: β-site APP cleaving enzyme 1; C99: Carboxy-terminal 99 amino acidfragment of APP generated by BACE1 cleavage; sAPPβ: Amino terminalectodomain fragment of APP generated by BACE1 cleavage.

Competing interestsR.V. has consulted for Eisai, Lilly, and Vitae Pharmaceutical Co.; his research fundingsources include NIH R01AG022560, R01AG030142, Cure Alzheimer’s Fund, BailaFoundation, BrightFocus Foundation, and the Alzheimer’s Association.

Authors’ contributionsRV planned the review, searched the primary sources of information, andwrote the review.

Authors’ informationRV is Professor of Cell and Molecular Biology at the Feinberg School of Medicine,Northwstern University, Chicago, IL, USA. He co-discovered BACE1 in 1999 andcurrently conducts research on the physiological functions of BACE1 and the roleof BACE1 in Alzheimer’s disease.

AcknowledgementsI thank Dr. Patty Kandalepas for making the figures. I also thank Drs. Bruce Albalaand Patrick May for reading and making useful comments on the Eisai and LillyBACE1 inhibitor clinical trial sections, respectively.

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